We hypothesised that the expression of selected proteases, matrix molecules, and collagens believed to have a role in the pathogenesis of OA would be changed in naturally occurring canin
Trang 1Open Access
Vol 8 No 6
Research article
Analysis of normal and osteoarthritic canine cartilage mRNA expression by quantitative polymerase chain reaction
Dylan N Clements1,2, Stuart D Carter1, John F Innes1, William ER Ollier2 and Philip JR Day2
1 The Musculoskeletal Research Group, c/o Department of Veterinary Pathology, Faculty of Veterinary Science, University of Liverpool, Liverpool, L69 3BX, UK
2 Centre for Integrated Genomic Medical Research, The Stopford Building, University of Manchester, Oxford Road, Manchester, M13 9PT, UK Corresponding author: Dylan N Clements, dylan.clements@liverpool.ac.uk
Received: 26 Apr 2006 Revisions requested: 18 May 2006 Revisions received: 7 Aug 2006 Accepted: 10 Oct 2006 Published: 10 Oct 2006
Arthritis Research & Therapy 2006, 8:R158 (doi:10.1186/ar2053)
This article is online at: http://arthritis-research.com/content/8/6/R158
© 2006 Clements et al.; licensee BioMed Central Ltd
This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
The molecular basis to mammalian osteoarthritis (OA) is
unknown We hypothesised that the expression of selected
proteases, matrix molecules, and collagens believed to have a
role in the pathogenesis of OA would be changed in naturally
occurring canine OA cartilage when compared to normal
articular cartilage Quantitative (real-time) reverse
transcriptase-polymerase chain reaction assays were designed measuring the
expression of selected matrix molecules (collagens and small
leucine-rich proteoglycans), key mediators of the proteolytic
degradation of articular cartilage (metalloproteinases,
cathepsins), and their inhibitors (tissue inhibitors of matrix
metalloproteinases) All data were normalised using a geometric
mean of three housekeeping genes, and the results subjected to
power calculations and corrections for multiple hypothesis
testing We detected increases in the expression of BGN,
COL1A2, COL2A1, COL3A1, COL5A1, CSPG2, CTSB, CTSD, LUM, MMP13, TIMP1, and TNC in naturally occurring
canine OA The expression of TIMP2 and TIMP4 was
significantly reduced in canine OA cartilage The patterns of gene expression change observed in naturally occurring canine
OA were similar to those reported in naturally occurring human
OA and experimental canine OA We conclude that the expression profiles of matrix-associated molecules in end-stage mammalian OA may be comparable but that the precise aetiologies of OA affecting specific joints in different species are presently unknown
Introduction
Osteoarthritis (OA) is the most common debilitating disease of
mammalian joints The clinical prevalence of human OA has
been estimated to affect 12.1% of the population aged 25 to
74 [1], whereas clinical OA affects up to 20% of the canine
population at large [2] Canine OA usually develops secondary
to an identifiable initiating cause (for example, secondary to hip
dysplasia [3]), although it can be experimentally induced [4]
Experimental models provide controlled and reproducible
development of OA [5], but only the study of naturally
occur-ring disease allows experimental findings to be directly related
to the clinical presentation with absolute certainty The related-ness of the pathogenesis of a common disease, such as OA,
in two different species has not been characterised [6]
At present, the precise mechanisms underlying the molecular pathogenesis of OA are unknown Quantification of gene expression is a fundamental tool for investigating gene func-tion in biological systems, particularly for elucidating patholog-ical mechanisms at play in diseased tissues Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) is currently considered the most accurate technique for quantify-ing gene expression With the publication of the canine genome [7], RT-PCR assays can now be readily designed for
ADAMTS5 = ADAM metallopeptidase with thrombospondin type 1 motif, 5; AGC1 = aggrecan; BGN = biglycan; CI = confidence interval; COL1A2
= type I collagen, alpha two chain; COL2A1 = type II collagen alpha 1 chain; COL3A1 = type III collagen alpha 1 chain; COL5A1 = type V collagen alpha 1 chain; COL9A3 = type IX collagen alpha 3 chain; CSPG2 = chondroitin sulphate proteoglycan 2; CT = mean threshold cycle; CTSB = cathe-psin B; CTSD = cathecathe-psin D; DCN = decorin; DF = degradation factor; ECM = extracellular matrix; FDR = false discovery rate; GAPDH = glyceral-dehyde-3-phosphate dehydrogenase; LUM = lumican; MMP = matrix metalloproteinase; OA = osteoarthritis; RIN = RNA integrity number; RPL13A
= ribosomal protein L13a; RT-PCR = reverse-transcriptase-polymerase chain reaction; SDHA = succinate dehydrogenase complex, subunit A; TBP
= TATA box binding protein; TIMP1 = tissue inhibitor of metalloproteinase 1; TIMP2 = tissue inhibitor of metalloproteinase 2; TIMP4 = tissue inhibitor
of metalloproteinase 4; TNC = tenascin C.
Trang 2the measurement of canine gene expression Although
canine-specific oligonucleotide microarrays are available for the
quan-tification of mRNA transcripts in canine tissue, such as
carti-lage [8], quantitative RT-PCR validation of the results
produced is still required
Articular cartilage is composed of chondrocytes embedded in
an extracellular matrix (ECM) The structural strength of the
matrix is provided by collagens such as type II collagen
(COL2), type VI collagen (COL6), type IX collagen (COL9),
type XI collagen (COL11), and type XVI collagen (COL16),
with COL2 accounting for 90% to 95% of the collagen
com-position of the ECM Other than water, the major
non-colla-genous component of articular cartilage is aggrecan (AGC1);
smaller components include the small leucine-rich
cans such as biglycan (BGN), chondroitin sulphate
proteogly-can 2 (CSPG2), decorin (DCN), lumiproteogly-can (LUM), and tenascin
C (TNC) The proteolytic degradation of normal and
osteoar-thritic cartilage matrix is performed by proteases such as the
matrix metalloproteinases (MMPs) [9], members of the
ADAMTS (a disintegrin and metalloproteinase with
throm-bospondin-like motif) family (or 'aggrecanases') [10], and
lys-osomal proteases (such as cathepsins) [11] Tissue inhibitors
of matrix metalloproteinases (TIMPs) are naturally occurring
inhibitors of MMP and ADAMTS function [12] The authors
are unaware of any publications documenting the change in
expression of structural ECM and protease collagens in the
articular cartilage of dogs with naturally occurring OA We
hypothesised that the expression of selected proteases, matrix
molecules, and collagens would be modulated in naturally
occurring canine OA
Materials and methods
Cartilage samples
Osteoarthritic articular cartilage was harvested from the
femo-ral heads of dogs that had end-stage natufemo-rally occurring OA
secondary to hip dysplasia (n = 15, mean age 2.7 years [range
1 to 12 years], mean weight 28.2 kg [range 25 to 36 kg]) and
that were undergoing routine surgical treatment of the disease
(total hip replacement) In all cases, severe clinical and
radio-graphic signs associated with OA of the affected joint
neces-sitated surgical treatment of the disease Articular cartilage
was harvested from the area surrounding the central cartilage
erosion usually observed on the canine OA hip [3] Normal
articular cartilage was harvested without visual evidence of hip
dysplasia or OA from the femoral heads of dogs, which had
been euthanatised for reasons unrelated to joint disease (n =
13, mean age 3.3 years [range 1 to 11 years], mean weight
26.2 kg [range 15 to 40 kg]) Articular cartilage was obtained
from the same site of the femoral head in the control dogs as
it was in diseased dogs Cartilage samples were immediately
immersed in RNAlater™ (Ambion Ltd., Huntingdon, UK) at
room temperature for 24 hours before being stored at -20°C
until use, in accordance with the manufacturer's instructions
RNA extraction from articular cartilage
Tissue samples were removed from RNAlater™ and total RNA was extracted using phenol/guanidine HCl reagents (Trizol™; Invitrogen Ltd, Paisley, UK) and isolated as previously described [13,14] An on-column DNA digestion step was included (RNase-Free DNase Set; Qiagen Ltd, Crawley, UK) Final elution of the total RNA was performed using 30 μl of RNase-free water and repeated to maximise the amount of RNA eluted
RNA quality assessment
The concentration of total RNA of each sample was quantified
by using a spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) RNA integrity was analysed by evalu-ating the capillary electrophoresis trace (Agilent 2100 Bioan-alyser; Agilent Technologies, Santa Clara, CA, USA) of the sample by using the RNA integrity number (RIN) algorithm [15], degradation factor (DF) [16], and ribosomal peak ratio The sample was determined to have minimal or no loss of integrity (RIN >6.4 and/or DF <10 and/or a ribosomal ratio
>0.4) and thus deemed suitable for use in the following exper-iments in accordance with a previously developed quality algo-rithm [14]
Synthesis of cDNA
Each sample was normalised to a concentration of 20 μg/μl, using RNAse-free water, and reverse transcription was per-formed using 10 μl RNA (200 μg total RNA) with oligo-dT12–
18 and Superscript II reverse transcriptase (Invitrogen Ltd) After reverse transcription, the template was diluted with 500
μl RNase/DNase-free water cDNA was stored at -80°C until later use in quantitative PCR
Quantitative PCR
Transcript sequences were obtained from the canine genome database [17], with cross-reference to the National Center for Biotechnology Information (Bethesda, MD, USA) [18] Where possible, assays were designed in areas of sequence showing 100% homology between predicted and verified sequences Primer and probe sequences were designed using online design software [19] To enhance the probability of transcript-specific PCR, selected amplicon systems were designed so that the last six to seven bases of a 3' primer or the probe crossed an exon-exon boundary When this was not possible, the primers were designed to be hybridised on different exons, with an intronic sequence greater than 1,100 base pairs, to maintain specificity for mRNA Some assays could be designed within only a single exon, and thus a genomic DNA assay was also designed to determine whether genomic con-tamination was present BLAST (Basic Local Alignment Search Tool) searches were performed for all primer sequences to confirm gene specificity
Genes were selected for assay on the basis of their impor-tance to cartilage homeostasis or pathology as derived from a
Trang 3literature review of naturally occurring human OA and
experi-mental canine OA and from the results of a preliminary
canine-specific whole genome microarray study, using a small number
of samples Assays were designed for quantification of
expres-sion of five collagen genes (type I collagen, alpha 2 chain
[COL1A2], type II collagen alpha 1 chain [COL2A1], type III
collagen alpha 1 chain [COL3A1], type V collagen alpha 1
chain [COL5A1], and type IX collagen alpha 3 chain
[COL9A3]), six ECM genes (AGC1, BGN, CSPG2, DCN,
LUM, and TNC), an intermediate filament (vimentin), proteases
and their inhibitors (ADAMTS-5, cathepsins B [CTSB],
cathe-psin D [CTSD], MMP13, TIMP1, TIMP2, and TIMP4), and
genomic DNA Assays for four reference genes
(glyceralde-hyde-3-phosphate dehydrogenase [GAPDH], TATA box
bind-ing protein [TBP], ribosomal protein L13a [RPL13A], and succinate dehydrogenase complex, subunit A [SDHA]) (Table
1) were also designed The reference genes used were selected from a panel of reference genes by applying a gene stability algorithm [20] Primers were synthesised by MWG Biotech (London, UK) Locked nucleic acid fluorescence res-onance energy transfer probes with a 5' reporter dye FAM (6-carboxy fluorescein) anda dark quencher dyewere synthesised
by Roche Diagnostics Ltd (Lewes, West Sussex, UK) The quantitative (real-time) PCR assays were all performed in triplicate using a TaqMan™ ABI PRISM 7900 SDS (Applied Biosystems, Foster City, CA, USA) in 384-well plate format Each assay well had a 10-μl reaction volume consisting of 5 μl
Table 1
A list of primer and probe sequences for the genes evaluated
ADAMTS5 TGGGTTCCCAAATATGCAG CTGTCCCATCCGTCACCT CTGGGAGA
1AGC1 GGGACCTGTGTGAGATCGAC GTAACAGTGGCCCTGGAACT AGGAGCTG
COL1A2 CTATCAATGGTGGTACCCAGTTT TGTTTTGAGAGGCATGGTTG GCCTGCTG
COL2A1 CTGGTGAACCTGGACGAGAG ACCACGATCACCCTTGACTC CCTCCTGG
COL3A1 GGATGGTGGCTTCCAGTTT CCAGCTGGACATCGAGGA GCTGCCTG
COL5A1 AACCTGTCGGATGGCAAGT CAGTCCAAGATCAAGGTGACAT CAGCATCC
COL9A3 CGAGGTGCCTCAGGTGAC ACCCAGCTCTCCTTTGTCC GAGACCAG
CSPG2 TGGATGGTTTTAATACGTTCAGG GCCGTAGTCACACGTCTCTG CTGCCTTC
GAPDH CTGGGGCTCACTTGAAAGG CAAACATGGGGGCATCAG CTGCTCCT
Genomic AACCCTCAAAGATGAGGTTTAGC ACTCTGGGATCACGCATGT CTGCCTTC
MMP13 CCGCGACCTTATCTTCATCT AACCTTCCAGAATGTCATAACCA AGAGGCAG
RPL13A CTGCCCCACAAGACCAAG GGGATCCCATCAAACACCT CCAGGCTG
TIMP1 TGCATCCTGCTGTTGCTG AACTTGGCCCTGATGACG CCCAGCAG
TIMP2 ATGGGCTGTGAGTGCAAGAT CACTCATCCGGAGACGAGAT CTGCCCCA
TIMP4 GCAGAGAGAAAGTCTGAATCATCA GGCACTGTATAGCAGGTGGTAA TGTGGCTG
ADAMTS5, ADAM metallopeptidase with thrombospondin type 1 motif, 5; AGC1, aggrecan; BGN, biglycan; COL1A2, type I collagen, alpha two
chain; COL2A1, type II collagen alpha 1 chain; COL3A1, type III collagen alpha 1 chain; COL5A1, type V collagen alpha 1 chain; COL9A3, type
IX collagen alpha 3 chain; CSPG2, chondroitin sulphate proteoglycan 2; CTSB, cathepsin B; CTSD, cathepsin D; DCN, decorin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Genomic, Genomic DNA, LUM, lumican;MMP13, matrix metalloproteinase 13; RPL13A, ribosomal protein L13a; SDHA, succinate dehydrogenase complex, subunit A; TBP, TATA box binding protein; TIMP1, tissue inhibitor of metalloproteinase 1; TIMP2, tissue inhibitor of metalloproteinase 2; TIMP4, tissue inhibitor of metalloproteinase 4; TNC, Tenascin C; VIM, vimentin.
Trang 42X PCR master mix with Uracil N-Glycosylase (Universal PCR
Mastermix; Applied Biosystems), 0.1 μl each of 20 μM forward
and reverse primers, 0.1 μl of 10 μM probe (Exiqon; Roche
Diagnostics Ltd), and 4.7 μl of sample cDNA (templates) or
water (negative controls)
The amplification was performed according to standard
proto-col with 10 minutes at 50°C followed by 40 cycles of 95°C for
1 minute and 60°C for 15 seconds, as recommended by the
manufacturer (Applied Biosystems) Real-time data were
ana-lysed by using the Sequence Detection Systems software,
version 2.2.1 (Applied Biosystems) The detection threshold
was set manually at 0.05 for all assays Standard curves were
generated for each assay (Additional file 1), to confirm that all assays were generated within acceptable limits (efficiency 93% > x > 107.4%) and R2 values (R2 > 0.98) (with the excep-tion of the genomic contaminaexcep-tion assay, in which efficiency was lower, but the detection of any transcript was deemed unacceptable)
Data analysis
The weights and ages of the patients were normally distributed
and thus compared with the calculation of means and Student t
tests The weight of the articular cartilage samples and quantity of RNA extract were compared using median values and
Mann-Whitney U tests because the data were not normally distributed.
Table 2
The dynamic range, standard curve slope, R 2 value, and efficiency of each polymerase chain reaction assay
Assay Lower detection limit (CT value) Upper detection limit (CT value) Standard curve slope R 2 value Efficiency
See Additional file 1 for further details ADAMTS5, ADAM metallopeptidase with thrombospondin type 1 motif, 5; AGC1, aggrecan; BGN, biglycan; COL1A2, type I collagen, alpha two chain; COL2A1, type II collagen alpha 1 chain; COL3A1, type III collagen alpha 1 chain; COL5A1, type V collagen alpha 1 chain; COL9A3, type IX collagen alpha 3 chain; CSPG2, chondroitin sulphate proteoglycan 2; CTSB, cathepsin B;
CTSD, cathepsin D; DCN, decorin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LUM, lumican;MMP13, matrix metalloproteinase 13; RPL13A, ribosomal protein L13a; SDHA, succinate dehydrogenase complex, subunit A; TBP, TATA box binding protein; TIMP1, tissue inhibitor
of metalloproteinase 1; TIMP2, tissue inhibitor of metalloproteinase 2; TIMP4, tissue inhibitor of metalloproteinase 4; TNC, Tenascin C; VIM,
vimentin.
Trang 5Real-time data were analysed by generation of mean threshold
cycle (CT) values from each transcript in triplicate Geometric
means [20] were calculated for the combined three reference
genes (GAPDH, TBP, and RPL13A) and used to calculate the
ΔΔCT (delta-delta CT) values and the relative amount of each
target gene [21] (Table 2) A fourth reference gene (SDHA)
was not included as a reference gene, because it was found
to have differential expression between normal and OA
sam-ples, even when included as part of the normalisation
calcula-tion The upper detection limit of dynamic range generated
from the standard curves was used as a cut-off point, above
which real-time data were discarded (that is, included in the
statistical analyses as zero/no transcript present)
Data were compared with the calculations of means, standard deviations, and fold changes from normal and paired
two-tailed t tests (body weight and age) performed in a
spread-sheet program (Microsoft Excel 2003; Microsoft Corporation, Redmond, WA, USA) and the calculation of graphs, 95%
con-fidence intervals (CIs) of the mean, and Mann-Whitney U tests
(to compare the amount of each target) performed in a statis-tical analysis software package (Minitab version 14.1; Minitab Ltd., Coventry, UK) One-sided power calculations were per-formed, assuming normality from the two samples with une-qual variance and using a freely available web-based program
[22] Significance was established at p < 0.05, and a robust
statistical analysis was assumed to have a power value greater than or equal to 80% Data were checked for errors due to
Table 3
Change in gene expression, mean 2 - ΔΔCT values, significance and power of comparisons between normal and OA canine articular cartilage
Gene Number of samples in which expression was
detected
2 -ΔΔCT normal 2-ΔΔCT OA Fold change in expression
(diseased versus normal)
Mann-Whitney U test p value
Power
TIMP4 27 0.109 0.043 -0.608 0.0094 0.859
TIMP2 28 3.959 1.664 -0.580 0.0020 0.844
ADAMTS5 16 0.031 0.019 -0.551 0.8478 0.175
COL3A1 26 10.573 37.867 2.581 0.0011 0.982
COL2A1 27 1.412 23.583 15.705 0.0001 0.779
ADAMTS5, ADAM metallopeptidase with thrombospondin type 1 motif, 5; AGC, aggrecan; BGN, biglycan; COL1A2, type I collagen, alpha two
chain; COL2A1, type II collagen alpha 1 chain; COL3A1, type III collagen alpha 1 chain; COL5A1, type V collagen alpha 1 chain; COL9A3, type
IX collagen alpha 3 chain; CSPG2, chondroitin sulphate proteoglycan 2; CTSB, cathepsin B; CTSD, cathepsin D; DCN, decorin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LUM, lumican; MMP13, matrix metalloproteinase 13; OA, osteoarthritis; RPL13A, ribosomal protein L13a; SDHA, succinate dehydrogenase complex, subunit A; TBP, TATA box binding protein; TIMP1, tissue inhibitor of metalloproteinase 1;
TIMP2, tissue inhibitor of metalloproteinase 2; TIMP4, tissue inhibitor of metalloproteinase 4; TNC, Tenascin C; VIM, vimentin.
Trang 6multiple hypothesis testing by using the Benjamini and
Hoch-berg false discovery rate (FDR) [23]
Results
There were no significant differences between the ages (mean
control 3.3 years [± 3.2 years, range 1 to 12 years], mean OA
2.7 years [± 3.1 years, range 1 to 11 years], p = 0.768) or
body weights (mean control 26.2 kg [± 8.0 kg, range 15 to 32
kg], mean OA 28.3 kg [± 3.8 kg, range 23 to 36 kg], p =
0.109) of the dogs in the diseased and control groups There
was no significant difference between the weight of the
carti-lage samples (median control 103 mg [range 45 to 260 mg],
median OA 92 mg [range 40 to 192 mg], p = 0.817) or the
quantity of RNA extracted, as determined by
spectrophotome-ter (median control 35 ng/μl [range 26 to 339 ng/μl], median
OA 42 ng/μl [range 22 to 247 ng/μl], p = 0.788).
Expression values are presented in Table 3 Two genes were
determined to have significant downregulation (TIMP2 and
TIMP4) in canine OA cartilage One gene was determined to
be significantly downregulated (SDHA) but with a low power
value (72%); this gene was excluded after FDR correction
Ten genes were determined to be significantly upregulated in
the OA samples (BGN, COL3A1, COL5A1, CSPG2, CTSB,
CSTD, LUM, MMP13, TIMP1, and TNC) Furthermore, in OA,
three genes were determined to be upregulated (COL1A2,
COL2A1, and COL9A3) but with low power values (74%,
78%, and 63%, respectively) and one gene was excluded
after FDR correction (COL9A3).
No amplification of genomic DNA was observed for any of the samples The average standard deviation for the triplicates in each assay was 16.9% (range 7.3% to 37.9%), indicating that all assays were reproducible Eleven of the 2,592 data points were removed because they were assumed to be aberrant (markedly different from the other two values in the triplicate)
None of the 'no template' control wells (n = 864) revealed a
signal Fold gene expression changes are illustrated in Figures
1 and 2, with all data normalised to the mean of the control val-ues (with a fold change of 0 being no change, a fold change
of 1 meaning a doubling of expression, and a fold change of -0.5 meaning a halving of expression) Statistical and power calculations are reported in Table 3
Figure 1
Graph illustrating the means and 95% confidence intervals (CIs) of the gene expression profiles
Graph illustrating the means and 95% confidence intervals (CIs) of the gene expression profiles To normalise values, the mean of each control
group has been used to normalise and produce fold changes in expression The results of the COL9A3 transcript are omitted because the 95% CIs were very high *Significant difference ADAMTS5, ADAM metallopeptidase with thrombospondin type 1 motif, 5; AGC, aggrecan; COL9A3, type IX collagen alpha 3 chain; CTSB, cathepsin B; D, disease; DCN, decorin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; N, normal; RPL13A, ribosomal protein L13a; SDHA, succinate dehydrogenase complex, subunit A; TBP, TATA box binding protein; TIMP2, tissue inhibitor of metallopro-teinase 2; TIMP4, tissue inhibitor of metalloprometallopro-teinase 4; TNC, Tenascin C; VIM, vimentin.
Trang 7Quantitative (real-time) RT-PCR is the most sensitive
tech-nique for the determination of mRNA transcript number [24]
To maximise the precision of our data, we included only mRNA
samples that had been determined as being of high quality
(using an algorithm determined by previous work [15]),
because mRNA degradation can affect assay performance
[24] Assays were optimised within specific limits of efficiency,
and the dynamic range of each assay was determined, used,
and presented with the expression data Additionally, we
cor-rected our results for multiple hypothesis testing (reducing the
opportunity for making a statistical type II error) and present
power values, allowing an interpretation of the strength of
each significant up- or downregulation
If variables such as the methods of mRNA extraction, RNA
quality assessment, reverse transcription, assay design,
meas-urement of genomic contamination, standard curve data
gen-eration, reference gene selection, and data normalisation were
presented in the 'Materials and methods' and 'Results'
sec-tions of manuscripts using quantitative PCR, more appropriate
comparison of results between different studies could be
made The geometric mean of three reference (housekeeping)
genes was used in this study to reduce the variability
associ-ated with the use of a single reference gene Geometric mean
methodology has been validated as a more accurate
normali-sation technique than that using a single reference gene if the
reference genes are selected through the use of a stability
algorithm [21], although in this study one of the genes
identi-fied by the algorithm (SDHA) was not stably expressed (Table
3)
Gene expression varies with both the site of cartilage harvest [25] and the degree of cartilage degeneration [26] in the OA joint We attempted to minimise this variability by using end-stage OA, age- and weight-matched samples, and stringent RNA quality control A relatively high degree of heterogeneity (large 95% CIs) was observed in the level of gene expression measured from the clinical samples in this study, even existing between samples within the same group This may reflect dif-ferences in dog age and/or breeds or variation in the time from surgical removal to collection in the preservative fluid The analysis of additional samples or the phenotyping and selec-tion of samples through histological grading may have increased the statistical powers of each of these differences observed, as the severity of OA measured by histology (Mankin score) correlates with a reduction in the expression of
COL2 and AGC [27].
Cell culture-based biological systems provide a more control-led methodology for evaluating gene expression when
com-pared with in vivo tissue For example, increased cell numbers
can be obtained, breed and age factors can be eradicated, and the absence of ECM facilitates the extraction of higher quality of mRNA [16] This is particularly true for studies of smaller mammals such as the dog, in which clinical samples of osteoarthritic cartilage may be less than 100 mg in size How-ever, cell-based models may differ in both gene expression
Figure 2
Graph illustrating the means and 95% confidence intervals (CIs) of the gene expression profiles
Graph illustrating the means and 95% confidence intervals (CIs) of the gene expression profiles To normalise values, the mean of each control
group has been used to normalise and produce fold changes in expression The results of the COL9A3 transcript are omitted because the 95% CIs were very high *Significant difference BGN, biglycan; COL1A2, type I collagen, alpha two chain; COL2A1, type II collagen alpha 1 chain;
COL3A1, type III collagen alpha 1 chain; COL5A1, type V collagen alpha 1 chain; COL9A3, type IX collagen alpha 3 chain; CSPG2, chondroitin
sulphate proteoglycan 2; CTSD, cathepsin D; D, disease; LUM, lumican; MMP13, matrix metalloproteinase 13; N, normal; TIMP1, tissue inhibitor of
metalloproteinase 1.
Trang 8profiles [28] or cell phenotype [29] with in vitro tissue
Ulti-mately, our understanding of the molecular pathogenesis of
OA requires relating changes observed with in vitro
experi-mentation to those identified from clinical tissue
The paucity of literature reporting changes in gene expression
observed in naturally occurring canine OA implies that often
this is not easy to achieve In part, this reflects the difficulties
associated with the use of clinical tissue samples, as noted
above, and the fact that the technology required to enable the
economic evaluation of gene expression across large groups
of tissue samples is only just becoming available Indeed, we
were limited by sample quantity, quality, and cost and needed
to rationalise our list of genes selected for evaluation, as
dis-cussed previously
We document marked elevation of expression in genes
encod-ing for collagen synthesis in the articular cartilage of dogs with
end-stage OA, which concurs with the findings in early
exper-imental canine OA [30-33] COL1A2, COL3A1, and
COL5A1 are characteristically synthesised by cells with a
fibrocartilaginous phenotype [34]
The increased expression of BGN, CSPG2, CTSB, DCN,
LUM, MMP13, and TNC is consistent with previous studies of
expression of these genes in both naturally occurring human
[35-39] and experimental canine OA [30-32] The biological
significance of fold changes in gene expression between
con-trol and OA samples is unknown in the absence of additional
data such as gross, radiographic, or histological scoring or
protein quantification Likewise, the changes in gene
expres-sion documented do not specify whether these changes are
causal or simply associated with the development of pathology
in the OA joint
We documented decreases in the expression of TIMP2 and
TIMP4 and an increase in the expression of TIMP1 in canine
OA cartilage The decrease in TIMP4 expression was
consist-ent with expression profiles of human OA cartilage [39],
although TIMP1 expression has been documented as being
decreased and TIMP2 expression has been documented as
being unchanged in human OA [39] Direct comparison of
gene expression levels with those measured in other joints
and/or in different species may be of limited value because the
underlying aetiologies to the development of OA may differ
However, the evaluation of structural matrix components and
proteases affecting those components is still of interest
because the end-stage pathology characterising canine OA
mimics that described for human OA [40]
Conclusion
On the basis of the results we present, the gene expression of
selected matrix molecules and key mediators of the proteolytic
degradation of articular cartilage is changed in end-stage,
nat-urally occurring OA of the canine hip The patterns of gene
expression change are broadly similar to those reported in experimental canine stifle OA and naturally occurring human OA
Competing interests
The Universal Probe Library™ was supplied at a reduced cost
by Roche Diagnostics Ltd
Authors' contributions
DNC collected and processed samples, carried out the molec-ular genetic studies, performed the statistical analysis, and drafted the manuscript SDC, JFI, and WERO conceived of the study, participated in its design and coordination, and helped to draft the manuscript PJRD participated in the design of the study, the assay design, and statistical analysis All authors read and approved the final manuscript
Additional files
Acknowledgements
Fiona Salway assisted in the design of the molecular genetic studies Malcolm Ness, Malcolm McKey, Toby Gemmill, Carlos Macias, Andrew Coughlan, and Christopher May contributed through the collection of samples DNC is funded by the Biotechnology and Biological Sciences Research Council, and the project was funded by the Petplan Charitable Trust.
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The following Additional files are available online:
Additional file 1
Standard curves generated for each assay by ten fold serial dilutions of template,withreal-time data analysed by using the Sequence Detection Systems software, version 2.2.1 (Applied Biosystems)
See http://www.biomedcentral.com/content/
supplementary/ar2053-S1.PDF
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